The major goal of mitosis is to distribute the genetic material accurately between the two daughter cells. Defects in meiosis or mitosis lead to aneuploidy, which is a significant cause of birth defects and is a hallmark of tumorigenesis. Proper spindle function relies on precise spatial and temporal control of microtubule (MT) dynamics and the integration of forces of motor proteins. Defects in regulated MT dynamics lead to spindle multi-polarity, improper kinetochore-MT attachments, delayed mitotic progression, and improper chromosome segregation. Despite the generation of an extensive parts list for the spindle, a major unanswered question is to understand how MT dynamics and motor protein activity are spatially and temporally regulated to ensure proper spindle architecture and chromosome segregation. Our lab?s work has been instrumental in defining how members of the kinesin superfamily contribute to spindle organization, chromosome congression, kinetochore-MT attachments, error correction, chromosome segregation, and cytokinesis. Over the course of this funded research, we proposed to study three major areas: 1) Examining mechanisms of motor regulation to integrate protein activity with its spatial and temporal control in the context of the spindle. 2) To visualize when and where individual motors are activated and to understand their interactions with key regulatory molecules. 3) To understand how defects in spindle architecture under increased chromosome load affect the fidelity of chromosome segregation. To understand the molecular mechanisms by which MT regulatory proteins and motors act during cell division, we often rely on time-lapse imaging to understand how cells divide, the rates of chromosome alignment and segregation, the fidelity of mitotic divisions, the overall rates of cell proliferation and the dynamics of cell migration. I am requesting a BioTek Lionheart FX multi-mode automated microscope. This microscope will be equipped with a live-cell imaging chamber for long-term time-lapse imaging of cultured cells in multi-well dishes. The system will be equipped with both phase and fluorescence optics. In addition, the system will include software for rapid analysis of cell proliferation, cell migration and sub-population analysis. The proposed new instrumentation will allow us to accelerate multiple routine assays and analyses, as well as open up new areas of investigation elucidating how cells divide and respond to drugs. In addition, by saving time on routine assays, we can devote more time to elucidating the molecular mechanisms of these processes, which will ultimately lead to a better understanding of how cytoskeletal targeted drugs act as effective therapeutic agents.